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1.
Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function.  相似文献   

2.
Wei Hou H  Gan HY  Bhagat AA  Li LD  Lim CT  Han J 《Biomicrofluidics》2012,6(2):24115-2411513
Sepsis is an adverse systemic inflammatory response caused by microbial infection in blood. This paper reports a simple microfluidic approach for intrinsic, non-specific removal of both microbes and inflammatory cellular components (platelets and leukocytes) from whole blood, inspired by the invivo phenomenon of leukocyte margination. As blood flows through a narrow microchannel (20 × 20 µm), deformable red blood cells (RBCs) migrate axially to the channel centre, resulting in margination of other cell types (bacteria, platelets, and leukocytes) towards the channel sides. By using a simple cascaded channel design, the blood samples undergo a 2-stage bacteria removal in a single pass through the device, thereby allowing higher bacterial removal efficiency. As an application for sepsis treatment, we demonstrated separation of Escherichia coli and Saccharomyces cerevisiae spiked into whole blood, achieving high removal efficiencies of ∼80% and ∼90%, respectively. Inflammatory cellular components were also depleted by >80% in the filtered blood samples which could help to modulate the host inflammatory response and potentially serve as a blood cleansing method for sepsis treatment. The developed technique offers significant advantages including high throughput (∼1 ml/h per channel) and label-free separation which allows non-specific removal of any blood-borne pathogens (bacteria and fungi). The continuous processing and collection mode could potentially enable the return of filtered blood back to the patient directly, similar to a simple and complete dialysis circuit setup. Lastly, we designed and tested a larger filtration device consisting of 6 channels in parallel (∼6 ml/h) and obtained similar filtration performances. Further multiplexing is possible by increasing channel parallelization or device stacking to achieve higher throughput comparable to convectional blood dialysis systems used in clinical settings.  相似文献   

3.
Organization of airway epithelium determines ciliary beat direction and coordination for proper mucociliary clearance. Fluidic shear stresses have the potential to influence ciliary organization. Here, an in vitro fluidic flow system was developed for inducing long-term airflow shear stresses on airway epithelium with a view to influencing epithelial organization. Our system consists of a fluidic device for cell culture, integrated into a humidified airflow circuit. The fluidic device has a modular design and is made from a combination of polystyrene and adhesive components incorporated into a 6-well filter membrane insert. We demonstrate the system operates within physiologically relevant shear and pressure ranges and estimate the shear stress exerted on the epithelial cell layer as a result of air flow using a computational model. For both the bronchial epithelial cell line BEAS2B and primary human tracheal airway epithelial cells, we demonstrate that cells remain viable within the device when exposed to airflow for 24 h and that normal differentiation and cilia formation occurs. Furthermore, we demonstrate the utility of our device for exploring the impact of exposing cells to airflow: our tool enables quantification of cytoskeletal organization, and is compatible with in situ bead assays to assess the orientation of cilia beating.  相似文献   

4.
In this paper, we develop a microfluidic device capable of generating nitric oxide (NO) gradients for cell culture using spatially controlled chemical reactions. NO plays an essential role in various biological activities, including nervous, immune, and cardiovascular systems. The device developed in this paper can control NO gradients without utilizing expensive and hazardous high purity NO gas sources or direct addition of NO donors. Consequently, the device provides an efficient, cost-effective, robust, and stable platform to generate NO gradients for cell culture studies. In the experiments, NO gradients are first characterized using a NO-sensitive fluorescence dye, and cell experiments using aortic smooth muscle cells are conducted. The results demonstrate that the device can alter the intracellular NO concentrations and further affect the Ca2+ concentration oscillation for the cells. The device developed in this paper provides a powerful platform for researchers better study the biological roles of NO and its spatial distribution using in vitro cell models with minimal instrumentation.  相似文献   

5.
We have designed, built, and evaluated a microfluidic device that uses deterministic lateral displacement for size-based separation. The device achieves almost 100% purity and recovery in continuously sorting two, four, and six micrometer microspheres. We have applied this highly efficient device to the purification of fungal (Aspergillus) spores that are spherical (∼4 μm diameter) with a narrow size distribution. Such separation directly from culture using unfiltered A. niger suspensions is difficult due to a high level of debris. The device produces a two to three increase in the ratio of spores to debris as measured by light scatter in a flow cytometer. The procedure is feasible at densities up to 4.4×106 spores∕ml. This is one of the first studies to apply microfluidic techniques to spore separations and has demonstrated that a passive separation system could significantly reduce the amount of debris in a suspension of fungal spores with virtually no loss of spore material.  相似文献   

6.
Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).  相似文献   

7.
We developed a microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM). We cocultured human umbilical vein endothelial cells (HUVECs) within the spheroids formed by hepatocellular carcinoma cells, and studied the distributions of the HUVECs over time. We observed that the migration of HUVECs depended on the size of spheroids. In the spheroids of ∼200 μm diameters, HUVECs migrated outwards to the edges within 48 h; while in the spheroids of ∼250 μm diameters, there was no outward migration of the HUVECs up to 72 h. In addition, we studied the effects of pro-angiogenic factors, namely, vascular endothelial growth factor (VEGF) and fibroblast growth factor (β-FGF), on the migration of HUVECs in the carcinoma cell spheroid. The outward migration of HUVECs in 200 μm spheroids was hindered by the treatment with VEGF and β-FGF. Moreover, some of the HUVECs formed hollow lumen within 72 h under VEGF and β-FGF treatment. The combination of SPIM and microfluidic devices gives high resolution in both spatial and temporal domains. The observation of HUVECs in spheroids provides us insight on tumor vascularization, an ideal disease model for drug screening and fundamental studies.  相似文献   

8.
Cui S  Liu Y  Wang W  Sun Y  Fan Y 《Biomicrofluidics》2011,5(3):32003-320038
This paper examined the feasibility of a microfluidics chip for cell capturing and pairing with a high efficiency. The chip was fabricated by the polydimethylsiloxane-based soft-lithography technique and contained two suction duct arrays set in parallel on both sides of a main microchannel. Cells were captured and paired by activating two sets of suction ducts one by one with the help of syringe pumps along with switching the cell suspensions inside the main microchannel correspondingly. The effects of suction flow rate and the dimensions of suction channels on the cell capturing and pairing efficiency were characterized. The present chip was capable of creating 1024 pairs of two different cell populations in parallel. The preliminary experimental results showed that the cell capturing efficiency was 100% and the pairing one was 88% with an optimal suction rate of 5 μl/min in the chip in the 2 μm-sized suction duct chip. The cell viability after capture inside the microfluidic device was 90.0 ± 5.3%. With this cell capturing and pairing chip, interaction between cells in a single pair mode can be studied. The ability to create cell pairs has a number of biological applications for cell fusion, cell-cell interaction studies, and cell toxicity screening.  相似文献   

9.
Huang SH  Hsueh HJ  Jiang YL 《Biomicrofluidics》2011,5(3):34109-3410910
This paper describes a light-addressable electrolytic system used to perform an electrodeposition of calcium alginate hydrogels using a digital micromirror device (DMD). In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-anode to electrolytically produce protons, which can lead to a decreased pH gradient. The low pH generated at the anode can locally release calcium ions from insoluble calcium carbonate (CaCO3) to cause gelation of calcium alginate through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressable electrodeposition of calcium alginate hydrogels with different shapes and sizes, as well as multiplexed micropatterning was performed. The effects of the concentration of the alginate and CaCO3 solutions on the dimensional resolution of alginate hydrogel formation were experimentally examined. A 3 × 3 array of cell-encapsulated alginate hydrogels was also successfully demonstrated through light-addressable electrodeposition. Our proposed method provides a programmable method for the spatiotemporally controllable assembly of cell populations into cellular microarrays and could have a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.  相似文献   

10.
Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.  相似文献   

11.
We have developed a method for studying cellular adhesion by using a custom-designed microfluidic device with parallel non-connected tapered channels. The design enables investigation of cellular responses to a large range of shear stress (ratio of 25) with a single input flow-rate. For each shear stress, a large number of cells are analyzed (500–1500 cells), providing statistically relevant data within a single experiment. Besides adhesion strength measurements, the microsystem presented in this paper enables in-depth analysis of cell detachment kinetics by real-time videomicroscopy. It offers the possibility to analyze adhesion-associated processes, such as migration or cell shape change, within the same experiment. To show the versatility of our device, we examined quantitatively cell adhesion by analyzing kinetics, adhesive strength and migration behaviour or cell shape modifications of the unicellular model cell organism Dictyostelium discoideum at 21 °C and of the human breast cancer cell line MDA-MB-231 at 37 °C. For both cell types, we found that the threshold stresses, which are necessary to detach the cells, follow lognormal distributions, and that the detachment process follows first order kinetics. In addition, for particular conditions’ cells are found to exhibit similar adhesion threshold stresses, but very different detachment kinetics, revealing the importance of dynamics analysis to fully describe cell adhesion. With its rapid implementation and potential for parallel sample processing, such microsystem offers a highly controllable platform for exploring cell adhesion characteristics in a large set of environmental conditions and cell types, and could have wide applications across cell biology, tissue engineering, and cell screening.  相似文献   

12.
Computational design optimization for microfluidic magnetophoresis   总被引：2，自引：0，他引：2  
Current macro- and microfluidic approaches for the isolation of mammalian cells are limited in both efficiency and purity. In order to design a robust platform for the enumeration of a target cell population, high collection efficiencies are required. Additionally, the ability to isolate pure populations with minimal biological perturbation and efficient off-chip recovery will enable subcellular analyses of these cells for applications in personalized medicine. Here, a rational design approach for a simple and efficient device that isolates target cell populations via magnetic tagging is presented. In this work, two magnetophoretic microfluidic device designs are described, with optimized dimensions and operating conditions determined from a force balance equation that considers two dominant and opposing driving forces exerted on a magnetic-particle-tagged cell, namely, magnetic and viscous drag. Quantitative design criteria for an electromagnetic field displacement-based approach are presented, wherein target cells labeled with commercial magnetic microparticles flowing in a central sample stream are shifted laterally into a collection stream. Furthermore, the final device design is constrained to fit on standard rectangular glass coverslip (60 (L)×24 (W)×0.15 (H) mm3) to accommodate small sample volume and point-of-care design considerations. The anticipated performance of the device is examined via a parametric analysis of several key variables within the model. It is observed that minimal currents (<500 mA) are required to generate magnetic fields sufficient to separate cells from the sample streams flowing at rate as high as 7 ml∕h, comparable to the performance of current state-of-the-art magnet-activated cell sorting systems currently used in clinical settings. Experimental validation of the presented model illustrates that a device designed according to the derived rational optimization can effectively isolate (∼100%) a magnetic-particle-tagged cell population from a homogeneous suspension even in a low abundance. Overall, this design analysis provides a rational basis to select the operating conditions, including chamber and wire geometry, flow rates, and applied currents, for a magnetic-microfluidic cell separation device.  相似文献   

13.
We present a low cost microfluidic chip integrating 3D micro-chambers for the capture and the analysis of cells. This device has a simple design and a small footprint. It allows the implementation of standard biological protocols in a chip format with low volume consumption. The manufacturing process relies on hot-embossing of cyclo olefin copolymer, allowing the development of a low cost and robust device. A 3D design of microchannels was used to induce high flow velocity contrasts in the device and provide a selective immobilization. In narrow distribution channels, the liquid velocity induces a shear stress that overcomes adhesion forces and prevents cell immobilization or clogging. In large 3D chambers, the liquid velocity drops down below the threshold for cell attachment. The devices can be operated in a large range of input pressures and can even be handled manually using simple syringe or micropipette. Even at high flow injection rates, the 3D structures protect the captured cell from shear stress. To validate the performances of our device, we implemented immuno-fluorescence labeling and Fluorescence in Situ Hybridization (FISH) analysis on cancer cell lines and on a patient pleural effusion sample. FISH is a Food and Drug Administration approved cancer diagnostic technique that provides quantitative information about gene and chromosome aberration at the single cell level. It is usually considered as a long and fastidious test in medical diagnosis. This process can be easily implanted in our platform, and high resolution fluorescence imaging can be performed with reduced time and computer intensiveness. These results demonstrate the potential of this chip as a low cost, robust, and versatile tool adapted to complex and demanding protocols for medical diagnosis.  相似文献   

14.
A porous silicon (PSi) based microarray has been integrated with a microfluidic system, as a proof of concept device for the optical monitoring of selective label-free DNA-DNA interaction. A 4 × 4 square matrix of PSi one dimensional photonic crystals, each one of 200 μm diameter and spaced by 600 μm, has been sealed by a polydimethylsiloxane (PDMS) channels circuit. The PSi optical microarray elements have been functionalized by DNA single strands after sealing: the microfluidic circuit allows to reduce significantly the biologicals and chemicals consumption, and also the incubation time with respect to a not integrated device. Theoretical calculations, based on finite element method, taking into account molecular interactions, are in good agreement with the experimental results, and the developed numerical model can be used for device optimization. The functionalization process and the interaction between DNA probe and target has been monitored by spectroscopic reflectometry for each PSi element in the microchannels.  相似文献   

15.
The angular resolution of an imaging device is the order of the reciprocal of the number of wavelengths across the aperture. To achieve the high resolution typical of optical instruments at microwaves, apertures miles in size may be required. The requirements for such a device, called a radio camera, are conformality with the terrain, a sparsely filled aperture of randomly (or nearly so) located elements, self-adaptive beam forming and a source of microwave illumination. Based on this design approach, a 1000-ft L-band radio camera is under development at the Valley Forge Research Center of the Moore School. This paper discusses the status of current development of the device.  相似文献   

16.
Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.  相似文献   

17.
A microfluidic device with planar square electrodes is developed for capturing particles from high conductivity media using negative dielectrophoresis (n-DEP). Specifically, Bacillus subtilis and Clostridium sporogenes spores, and polystyrene particles are tested in NaCl solution (0.05 and 0.225 S∕m), apple juice (0.225 S∕m), and milk (0.525 S∕m). Depending on the conductivity of the medium, the Joule heating produces electrothermal flow (ETF), which continuously circulates and transports the particles to the DEP capture sites. Combination of the ETF and n-DEP results in different particle capture efficiencies as a function of the conductivity. Utilizing 20 μm height DEP chambers, “almost complete” and rapid particle capture from lower conductivity (0.05 S∕m) medium is observed. Using DEP chambers above 150 μm in height, the onset of a global fluid motion for high conductivity media is observed. This motion enhances particle capture on the electrodes at the center of the DEP chamber. The n-DEP electrodes are designed to have well defined electric field minima, enabling sample concentration at 1000 distinct locations within the chip. The electrode design also facilitates integration of immunoassay and other surface sensors onto the particle capture sites for rapid detection of target micro-organisms in the future.  相似文献   

18.
Cancer cell migration through tissue pores and tracks into the bloodstream is a critical biological step for cancer metastasis. Although in vivo studies have shown that expression of vimentin can induce invasive cell lines, its role in cell cytoskeleton reorganization and cell motility under in vitro physical confinement remains unknown. Here, a microfluidic device with cell culture chamber and collagen-coated microchannels was developed as an in vitro model for physiological confinement environments. Using this microchannel assay, we demonstrated that the knockdown of vimentin decreases 3T3 fibroblast cell directional migration speed in confined microchannels. Additionally, as cells form dynamic membranes that define the leading edge of motile cells, different leading edge morphologies of 3T3 fibroblast and 3T3 vimentin knockdown cells were observed. The leading edge morphology change under confinement can be explained by the effect of vimentin on cytoskeletal organization and focal adhesion. The microfluidic device integrated with a time-lapse microscope provided a new approach to study the effect of vimentin on cell adhesion, migration, and invasiveness.  相似文献   

19.
A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.  相似文献   

20.
Bacterial culture is a basic technique in both fundamental and applied microbiology. The excessive reagent consumption and laborious maintenance of bulk bioreactors for microbial culture have prompted the development of miniaturized on-chip bioreactors. With the minimal choice of two compartments (N = 2) and discrete time, periodic dilution steps, we realize a microfluidic bioreactor that mimics macroscopic serial dilution transfer culture. This device supports automated, long-term microbial cultures with a nanoliter-scale working volume and real-time monitoring of microbial populations at single-cell resolution. Because of the high surface-to-volume ratio, the device also operates as an effective biofilm-flow reactor to support cogrowth of planktonic and biofilm populations. We expect that such devices will open opportunities in many fields of microbiology.  相似文献   

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